Marine biomass, such as large marine algae, is a promising starting material for biofuel. Examples of major reasons therefor include: (i) large marine algae exhibit higher productivity than terrestrial biomass; (ii) unavoidable problems that arise when cultivating terrestrial biomass (e.g., irrigation or dressing) can be avoided since cropland is not necessary; and (iii) large marine algae are lignin-free. Major examples of large marine algae include green algae, red algae, and brown algae. Among them, at least red algae and brown algae contain significant amounts of carbohydrates. Gelidium amansii, which is one type of red algae, contains 17% cellulose (glucose) (w/w (dry weight basis); hereafter, “w/w” refers to dry weight unless otherwise specified) and 58.6% (w/w) agar (25.6% galactose and 33% 3,6-anhydrogalactose). Brown algae contain 40% (w/w) alginic acid, 30% (w/w) mannitol, and 30% (w/w) laminarin at maximum. Therefore, biofuel production using large marine algae as starting materials requires the establishment of a technique for converting such carbohydrate components into biofuel.
Alginic acid is a linear acidic polysaccharide composed of β-D-mannuronic acid (M) and its C5-epimer (i.e., α-L-guluronic acid (G)). A constitutive monosaccharide has a poly-M, poly-G, or poly-MG structure. Mannitol is a sugar alcohol corresponding to mannose, and it is oxidized via the action of mannitol dehydrogenase and then converted into fructose (FIG. 1) (refer to Non-Patent Documents 1 and 2). Laminarin is composed of β-(1,3)-D-glucan having a 3-(1,6)-branching structure (refer to Non-Patent Documents 3 and 4). The present inventors have already constructed a system for producing ethanol from alginic acid using the ethanol-producing Sphingomonas sp. A1 strain (hereafter referred to as “the ethanol-producing A1 strain”) and succeeded in producing 1.3% (w/v) ethanol (refer to Non-Patent Document 5). Such technique is an only one technique for producing biofuel from alginic acid. There have been a few examples of ethanol production from laminarin. However, there has been a report regarding ethanol production via laminarin decomposition by three yeast strains (i.e., Kluyveromyces marxianus, Pacchysolen tannophilus, and Phicia angophorae) (refer to Non-Patent Document 1), as well as a report regarding ethanol production using a yeast strain (i.e., Saccharomyces cerevisiae) from a product of laminarin decomposed by a laminarin-degrading enzyme (i.e., laminarise) (Adams et al., 2009). Regarding production of ethanol from mannitol, it was reported that the bacterial strains (i.e., Zymobacter palmae and Escherichia coli KO11) had produced about 1.3% (w/v) and 2.6% (w/v) ethanol from 3.8% (w/v) and 9.0% (w/v) mannitol with production efficiency of 0.38 g and 0.41 g of ethanol (mannitol)−1, respectively (refer to Non-Patent Documents 1 and 6). From the viewpoint of ethanol production, however, yeast strains are considered to be advantageous over bacterial strains in various respects, such as tolerance to ethanol or fermentation inhibitors (refer to Non-Patent Document 7) (Hughes and Qureshi, 2010). In fact, Z. palmae and E. coli KO11 inhibit growth in the presence of 5% (w/v) ethanol (refer to Non-Patent Documents 8 and 9). However, there has been very little research regarding ethanol production from mannitol using yeast. Such research is limited to a report to the effect that a yeast strain (S. cerevisiae polyploid BB1) produces about 0.5% ethanol from 5% (w/v) mannitol (refer to Non-Patent Document 2) and a report to the effect that only P. angophorae among the laminarin-degrading yeast strains mentioned above (i.e., K. marxianus, P. tannophilus, and P. angophorae) produces about 1.0% (w/v) ethanol from 4% (w/v) mannitol with production efficiency of 0.40 g of ethanol (mannitol)−1 (refer to Non-Patent Document 1). In research involving the use of P. angophorae, ethanol production from an algal extract comprising both mannitol and laminarin and the influence of the amount of oxygen supplied on the speed of mannitol and laminarin consumption have been reported (refer to Non-Patent Document 1). However, only a few reports have been made regarding mannitol metabolism using yeast. It has been reported that yeast strains (S. cerevisiae) are classified as those capable of mannitol assimilation (e.g., the polyploid BB1 strain and the monoploid A184D strain) and those incapable of mannitol assimilation (or having a very weak capacity for assimilation, such as the polyploid BB2 strain and the haploid S288C and Sc41 YJO strains). It has also been reported that mannitol assimilation using S. cerevisiae requires oxygen and yeast strains growing in a mannitol-containing medium exhibit a high degree of respiratory activity (refer to Non-Patent Documents 2 and 10). The monoploid strain S288C is the first strain the genome sequence of which was determined (refer to Non-Patent Document 11).
In order to achieve practical use of ethanol production from mannitol using yeast, it is essential to search for yeast strains exhibiting a high degree of ethanol productivity from mannitol or various other excellent properties, or to breed such strains and to establish optimal conditions for exerting a high degree of ethanol productivity. In order to establish an ethanol production system from marine biomass, further, a technique for converting all of the constituents into ethanol is necessary. In the case of brown algae, it is necessary to establish a technique for converting alginic acid, mannitol, laminarin, and the like into ethanol. Known systems for production of ethanol from alginic acid are limited to a single system involving the use of the ethanol-producing A1 strain described above (refer to Non-Patent Document 5). The A1 strain is not capable of mannitol or laminarin assimilation (unpublished data). The capacity for alginic acid assimilation is known only in a limited number of organisms, such as the Sphingomonas sp. A1 strain.